ITP is an electrokinetic technique used to preconcentrate and separate ionic analytes (Everaerts et al., Isotachophoresis: Theory, Instrumentation, and Applications, Elsevier, Amsterdam, N.Y., 1976; Bocek, Analytical Isotachophoresis, VCH, Cambridge, 1987; Garcia-Schwarz et al. (2012) JoVE e3890). The ITP method involves introduction of a sample comprising an analyte of interest between a zone containing a “fast” migrating leading electrolyte (LE) and a zone containing a “slow” migrating trailing electrolyte (TE). The LE and TE are chosen such that the mobility of the analyte of interest is higher in the TE zone than that of the TE co-ion and lower in the LE zone than that of the LE co-ion. Analytes can be co-focused by this method into a relatively narrow peak at the interface of the LE and TE electrolytes (Garcia-Schwarz et al., supra; Khurana et al. (2008) Anal. Chem. 80:6300; Garcia-Schwarz et al. (2011) J. Fluid Mech. 679:455).
Affinity chromatography (AC) is a chromatographic technique that leverages a specific binding agent, the affinity ligand, for purification, separation, and/or analysis of sample components. The affinity ligand (probe) is used to selectively yet reversibly capture the sample component of interest (target). Numerous samples and sample components are analyzed or purified using AC, including enzymes, lectins, other proteins, and nucleic acids (Hage, Handbook of Affinity Chromatography, CRC Press, Boca Raton, 2006; Hage (1999) Clin. Chem. 45:593; Mallik et al. (2006) J. Sep. Sci. 29:1686; Pfaunmiller et al. (2013) Anal. Bioanal. Chem. 405:2133). For many samples, including important biological samples (e.g., blood, cell lysate), the component of interest is present in very low concentrations, while background, potentially fouling species are present in relatively high concentrations. This necessitates that a substantial volume of sample be processed through the affinity substrate. Additionally, low target concentrations imply low target-probe binding rates (Levenspiel, Chemical Reaction Engineering John Wiley & Sons, New York, 1999). These factors increase the time of the affinity assay, can lead to poor substrate utilization, and/or poor purification yield, limiting applications of the method. The aforementioned limitations of AC can be addressed by increasing the forward binding rate of reactions (Levenspiel, supra), but this binding rate is often difficult to improve upon (Hage, supra).
ITP has been used in conjunction with affinity assays in several applications. For example, Abelev et al. used ITP with counterflow to transport sample compounds onto regions of immobilized proteins on cellulose acetate and nitrocellulose membrane (Abelev et al. (1988) Bull. Exp. Biol. Med. 106:1600; Abelev et al. (1989) Mol. Immunol. 26:49; Abelev et al. (1989) Mol. Immunol. 26:41; Abelev et al. (1988) Bull. Exp. Biol. Med. 105:748; Schranz et al. (1991) Electrophoresis 12:414; Abelev and Karamova, in Serological Cancer Markers, Springer, 1992, p. 453; Abelev et al. (1994) Mol. Biol. (Mosk) 28:768). Abelev et al. used the binding to detect the presence of and analyze properties of antibodies, proteins, lectins, and nucleic acids. However, in their work, ITP was used only as a pump-free, reproducible transport mechanism, and not to substantially preconcentrate the analytes or speed up reactions (Abelev et al. (1994), supra). More recently, Garcia-Schwartz et al. presented an approach combining ITP and an affinity reaction to detect micro-RNA (Garcia-Schwarz et al. (2012) Anal. Chem. 84:6366; Garcia-Schwarz et al. (2013) Angew. Chem. 125:11748). They used ITP to accelerate hybridization between a target and a mobile fluorescent DNA probe in a microchannel. This ITP zone was then transported into a channel section containing cross-linked polyacrylamide gel functionalized with DNA complementary to the fluorescent DNA probe. This method was used to remove signal background (a negative enrichment strategy) and enhance sequence specific quantitation (Garcia-Schwarz et al. (2012), supra; Garcia-Schwarz et al. (2013), supra).
Microarray technology enables the investigation of on the order of 10,000 sequences in parallel (Lander (1999) Nat. Genet. 21:3-4). This high-throughput capability and sensitivity has been leveraged in a wide range of applications, including gene expression analysis (Ross et al. (2000) Nat. Genet. 24:227-235; Adomas et al. (2008) Tree Physiol. 28:885-897; Schena et al. (1995) Science 270:467-470), diagnosis of diseases (Marx (2000) Science 289:1670-1672; Wallace (1997) Mol. Med. Today 3:384-389; Shen et al. (2009) Clin. Chem. 55:659-669), single nucleotide polymorphism (SNP) (Hacia et al. (1999) Nat. Genet. 22:164-167; Chen et al. (2003) Pharmacogenomics J. 3:77-96), and aptamer-based analysis of protein biomarkers (Gold et al. (2010) PLoS One 5:e15004; Kraemer et al. (2011) PLoS One 6:e26332). DNA arrays use hybridization between a mixture of targets suspended in a bulk sample solution and probes immobilized on a solid substrate. Despite its proven success, application of DNA arrays to rapid screening of samples remains a challenge (Teles et al. (2008) Talanta 77:606-623). The hybridization alone in conventional microarray experiments typically requires overnight (15-24 hours) incubation to yield measurable signal from target molecules at a wide range of target concentrations (Hegde et al. (2000) J. Biotechniques 29:548-550, 552-554, 556 passim; Cheung et al. (1999) Nat. Genet. 21(1 Suppl):15-19). This constraint has limited the application of microarrays in point-of-care applications where short turnaround time is desirable. Reduction in hybridization time is also favorable because extended incubation times are associated with solution-dependent cleavage of the linkage chemistry between the probe and the solid support, negatively affecting the reproducibility and sensitivity (Situma et al. (2006) Biomol. Eng. 23:213-231).
There are two main challenges in speeding up hybridization processes: overcoming the slow diffusion-limited target transport and the slow reaction rates associated with low target concentrations (Pappaert et al. (2003) Chem. Eng. Sci. 58:4921-4930). The vast majority of work toward speeding up DNA array hybridization has involved addressing the first of these limitations with active pumping and/or mixing of liquid solution containing the molecular target (Wang et al. (2011) Anal. Chim. Acta 687:12-27). Successful microarray hybridization speed-up has been demonstrated using syringe pump-driven mixing (McQuain et al. (2004) Anal. Biochem. 325:215-226), microfluidic integrated peristaltic pump mixing (Liu et al. (2006) Angew Chem. Int. Ed. Engl. 45(22):3618-3623), mixing using 7 mm scale magnetic stir bars (Yuen et al. (2003) Lab Chip 3:46-50), pumping and mixing discrete sample plugs through serpentine microchannels (Wei et al. (2005) Nucleic Acids Res. 33:e78), acoustic microstreaming (Liu et al. (2003) Anal. Chem. 75:1911-1917), pumping with displacement micropumps, centrifugal liquid pumping (Wang et al. (2010) Anal. Biochem. 400:282-288; Peytavi et al. (2005) Clin. Chem. 51:1836-1844; Chen et al. (2008) Lab Chip 8:826-829), pneumatically driven mixing (Wang et al. (2011) J. Talanta 84:565-571; Adey et al. (2002) Anal. Chem. 74:6413-6417), and electrokinetic sample dispensing and washing (Erickson et al. (2004) Anal Chem. 76(24):7269-7277).
The aforementioned methods of active pumping and vigorous mixing help hybridization rate as they create conditions where fresh sample at its original concentration is driven toward ligands on the surface, avoiding diffusion limited regime. However, these approaches do not address the challenge of kinetically limited reactions associated with low target concentrations. Kinetically limited reaction between suspended DNA with surface-bound cDNA can take hours to reach equilibrium due to the inherent dynamics of bulk-to-surface reactions (Gao et al. (2006) Nucleic Acids Res. 34:3370-3377; Okahata et al. (1998) Anal. Chem. 70:1288-1296). Kinetically limited hybridization is therefore not addressed by pumping or mixing, but rather by preconcentration of target species or methods of increasing kinetic parameters themselves (e.g., varying temperature). An example of preconcentration-driven reaction rate enhancement is the work of Edman et al. (Nucleic Acids Res. (1997) 25:4907-4914), who demonstrated 30-fold increased hybridization rate by electrophoretically accumulating DNA species at the capture probes immobilized on the surface of positively biased microelectrodes. This method received much attention for a brief time, but was largely abandoned by the field, possibly due to its strict microfabrication requirements, and the strong sensitivity of hybridization reactions to the effects of electrochemical reactions (which can damage DNA, dramatically change local pH, and/or generate bubbles due to water hydrolysis).
Thus, there remains a need for methods of increasing the rates of kinetically limited ligand binding or nucleic acid hybridization reactions, particularly in affinity chromatography and microarray applications.